Novel pharmaceutical dosage forms provide for pulsatile delivery of methylphenidate, i.e., release encapsulated drug in spaced apart xe2x80x9cpulses.xe2x80x9d The dosage forms are comprised of first, second and optionally third dosage units, with each dosage unit having a different drug release profile. The dosage forms may comprise capsules housing compressed tablets or drug-containing beads or particles, or may comprise a single tablet with the first, second and optionally third dosage units each representing an integral and discrete segment thereof. Methods of treatment using the pharmaceutical dosage forms are provided as well.

The present invention relates generally to drug delivery, and more specifically relates to novel pharmaceutical dosage forms that provide pulsatile delivery of methylphenidate. The invention additionally relates to methods for administering methylphenidate using the novel dosage forms.
Pharmaceutical dosage forms are known which provide a variety of drug release profiles, including immediate release, sustained release, and delayed release. That is, it may be desirable, for a particular drug, to prevent drug release after drug administration until a certain amount of time has passed (so-called xe2x80x9ctimed releasexe2x80x9d), to provide substantially continuous release over a predetermined time period (so-called xe2x80x9csustained releasexe2x80x9d) or to provide release immediately following drug administration (i.e., xe2x80x9cimmediate releasexe2x80x9d). For some types of drugs, it is preferred to release the drug in xe2x80x9cpulses,xe2x80x9d wherein a single dosage form provides for an initial dose of drug followed by a release-free interval, after which a second dose of drug is released, followed by one or more additional release-free intervals and drug release xe2x80x9cpulses.xe2x80x9d Pulsatile drug delivery is useful, for example, with active agents that have short half-lives and must be administered two or three times daily, with active agents that are extensively metabolized presystemically, and with active agents which lose the desired therapeutic effect when constant blood levels are maintained. These types of agents have pharmacokinetic-pharmacodynamic relationships that are best described by a clockwise xe2x80x9chysteresis loop.xe2x80x9d A drug dosage form that provides a pulsatile drug release profile is also useful for minimizing the abuse potential of certain types of drugs, i.e., drugs for which tolerance, addiction and deliberate overdose can be problematic.
Because a precise and effective pulsatile drug delivery system is difficult to formulate and manufacture, there are few such dosage forms that have been commercialized. There are, however, several patents and literature references pertaining to pulsatile drug delivery. See, for example, U.S. Pat. No. 5,413,777 to Sheth et al., directed to a pulsatile once-a-day delivery system for the administration of minocycline; U.S. Pat. No. 5,260,068 to Chen, directed to a multiparticulate pulsatile drug delivery system; U.S. Pat. No. 4,777,049 to Magruder et al., directed to an osmotic delivery system for constant release of a drug with intermittent release xe2x80x9cpulsesxe2x80x9d; U.S. Pat. No. 5,391,381 to Wong et al., directed to a drug dispenser for delivering individual drug-containing units in a xe2x80x9cpulsatilexe2x80x9d manner; PCT Publication No. WO 98/32424, pertaining to pulsatile delivery of diltiazem hydrochloride; U.S. Pat. Nos. 5,472,708 and 5,260,069 to Chen; Ishino et al. (1992) xe2x80x9cDesign and Preparation of Pulsatile Release Tablet as a New Oral Drug Delivery System,xe2x80x9d Chem. Pharm. Bull. 40(11):3036-3041; Cohen et al. (1994), xe2x80x9cPulsatile Release from Microencapsulated Liposomes,xe2x80x9d J. Liposome Res. 349-360; and Gazzaniga et al. (1994), xe2x80x9cChronotopic Drug Delivery Systems for Pulsatile and/or Site-Specific Release,xe2x80x9d 21st. Proc. Int. Symp. Controlled Release Bioact. Mater., pp. 744-745.
The present invention is directed in part to a novel pulsatile drug delivery system which is straightforward to manufacture and provides precisely timed drug release xe2x80x9cpulsesxe2x80x9d at desired intervals.
Methylphenidate hydrochloride (HCl), the hydrochloride salt of xcex1-phenyl-2-piperidine-acetic acid methyl ester (available commercially as Ritalin(copyright)), is a central nervous system stimulant that is used in the treatment of Attention Deficit Disorder (xe2x80x9cADDxe2x80x9d), a commonly diagnosed nervous system illness in children that is characterized by both distractability and impulsivity. Methylphenidate HCl is also used to treat a related disorder, Attention Deficit Hyperactivity Disorder (xe2x80x9cADHDxe2x80x9d), in which symptoms of hyperactivity are present along with the symptoms of ADD. The drug is additionally used in the symptomatic treatment of narcolepsy, depression, and the cognitive decline associated with Acquired Immunodeficiency Syndrome (xe2x80x9cAIDSxe2x80x9d) or AIDS-related conditions, as well as for mood elevation, particularly in terminally ill patients with diseases such as cancer. Methylphenidate exists as four distinct isomers, as follows:
The drug as used in therapy is a racemic mixture of the d- and l-threo enantiomers, which have been acknowledged as more active than the erythro pair.
Because of its potential for tolerance (loss of clinical efficacy when constant blood levels are maintained), short-half life and potential for abuse, methylphenidate is a primary candidate for use in conjunction with the drug delivery systems of the invention.
Accordingly, the present invention provides novel pharmaceutical dosage forms for the administration of methylphenidate in its conventional form, i.e., as a racemic mixture of the d-threo and l-threo enantiomers. The novel dosage forms provide for pulsatile drug release, thereby maximizing efficacy (i.e., the loss of clinical efficacy over time), reducing the potential for abuse or noncompliance. The invention thus represents a significant advance in the art, particularly in the administration of drugs such as methylphenidate that have short half-lives, tend to be extensively metabolized presystemically, have pharmacokinetic-pharmacodynamic relationships which are best described by positive (clockwise) hysteresis loops (hence resulting in tolerance when constant blood levels are maintained), or are likely candidates for drug abuse. No art of which applicants are aware describes pulsatile drug delivery systems as now provided herein.
To the best of applicants"" knowledge, the pharmaceutical dosage forms of the invention are previously unknown and completely unsuggested by the art.
Accordingly, it is a primary object of the invention to address the above-mentioned need in the art by providing a pharmaceutical dosage form for pulsatile delivery of methylphenidate.
It is another object of the invention to provide such a dosage form comprising at least two individual drug-containing dosage units, each of which has a different drug release profile.
It is another object of the invention to provide such a dosage form wherein the dosage units are housed in a closed capsule.
It is still another object of the invention to provide such a dosage form wherein the dosage units are compressed tablets.
It is yet another object of the invention to provide such a dosage form wherein the dosage units are drug-containing particles or beads.
It is a further object of the invention to provide such a dosage form comprised of a single tablet of which the drug-containing dosage units represent integral but discrete segments.
It is a further object of the invention to provide such a dosage form for administering methylphenidate optionally in combination with one or more other active agents such as CNS stimulants (including analeptic agents and psychostimulants), antidepressant drugs, antianxiety agents and the like.
It is an additional object of the invention to provide methods for administering methylphenidate using the novel dosage forms.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
Definitions and Nomenclature
Before the present formulations and methods of use are disclosed and described, it is to be understood that unless otherwise indicated this invention is not limited to specific pharmacologically active agents, specific pharmaceutical carriers, or to particular administration regimens, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
It must be noted that, as used in the specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9can active agentxe2x80x9d includes mixtures of active agents, reference to xe2x80x9ca pharmaceutical carrierxe2x80x9d includes combinations of two or more carriers, and the like.
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
xe2x80x9cOptionalxe2x80x9d or xe2x80x9coptionallyxe2x80x9d means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
The terms xe2x80x9cactive agent,xe2x80x9d xe2x80x9cdrugxe2x80x9d and xe2x80x9cpharmacologically active agentxe2x80x9d are used interchangeably herein to refer to a chemical material or compound which, when administered to an organism (human or animal, generally human) induces a desired pharmacologic effect. In the context of the present invention, the terms refer to a compound that is capable of being delivered orally.
The term xe2x80x9cmethylphenidatexe2x80x9d as used herein refers to a racemic mixture of d-threo methylphenidate and l-threo methylphenidate, also referred to herein as xe2x80x9cd,l-methylphenidate.xe2x80x9d
By the terms xe2x80x9ceffective amountxe2x80x9d or xe2x80x9cpharmaceutically effective amountxe2x80x9d of an agent as provided herein are meant a nontoxic but sufficient amount of the agent to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, and the particular active agent administered, and the like. Thus, it is not possible to specify an exact xe2x80x9ceffective amount.xe2x80x9d However, an appropriate xe2x80x9ceffectivexe2x80x9d amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
By xe2x80x9cpharmaceutically acceptablexe2x80x9d carrier is meant a carrier comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected active agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The term xe2x80x9ccarrierxe2x80x9d is used generically herein to refer to any components present in the pharmaceutical formulations other than the active agent or agents, and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
Similarly, a xe2x80x9cpharmaceutically acceptablexe2x80x9d salt or a xe2x80x9cpharmaceutically acceptablexe2x80x9d ester of a compound as provided herein is a salt or ester which is not biologically or otherwise undesirable.
In the chemical structures drawn herein, the use of bold and dashed lines to denote particular conformation of substituents follows IUPAC convention. The symbols xe2x80x9cxcex1xe2x80x9d and xe2x80x9cxcex2xe2x80x9d indicate the specific stereochemical configuration of a substituent at an asymmetric carbon atom in a chemical structure as drawn. Thus xe2x80x9cxcex1,xe2x80x9d denoted by a broken line, indicates that the group in question is below the general plane of the molecule as drawn, and xe2x80x9cxcex2,xe2x80x9d denoted by a bold line, indicates that the group at the position in question is above the general plane of the molecule as drawn.
xe2x80x9cPulsatile Releasexe2x80x9d Dosage Forms
In a first embodiment, the invention features pharmaceutical dosage forms that provide for pulsatile delivery of methylphenidate. By xe2x80x9cpulsatilexe2x80x9d is meant that a plurality of drug doses are released at spaced apart time intervals. Generally, upon ingestion of the dosage form, release of the initial dose is substantially immediate, i.e., the first drug release xe2x80x9cpulsexe2x80x9d occurs within 1-2 hours of ingestion. This initial pulse is followed by a first time interval during which substantially no drug is released from the dosage form, after which a second dose is then released. Typically, the second dose is released on the order of 3-5 hours following ingestion of the dosage form. Preferably, release of the second dose is followed by a second non-release interval, which is again followed by a xe2x80x9cpulsexe2x80x9d of drug release. Ideally, release of a third dose occurs on the order of 7-9 hours following ingestion. In a preferred embodiment herein, either two or three release pulses are provided. However, the invention is also intended to encompass dosage forms that provide more than three pulses, with non-release intervals therebetween of approximately 2-6 hours, preferably 3-5 hours.
The aforementioned pulsatile release profile is achieved with dosage forms that, in one embodiment, are closed and preferably sealed capsules housing two or more drug-containing xe2x80x9cdosage units.xe2x80x9d In a preferred embodiment, each dosage unit comprises a compressed or molded tablet, wherein each of the tablets within the capsule provides a different drug release profile. That is, for an exemplary dosage form, a first tablet releases drug substantially immediately following ingestion of the dosage form, while a second tablet in the capsule releases drug approximately 3-5 hours following ingestion, and an optional third tablet provides drug release after approximately 7-9 hours. While the dosage form will not generally include more than three tablets, dosage forms housing four or more tablets are within the scope of the present invention.
In an alternative embodiment, each dosage unit comprises a drug-containing particle or bead (drug-containing xe2x80x9cbeadsxe2x80x9d refer to drug-coated inert supports, e.g., lactose beads coated with drug). A first group of these particles or beads releases drug substantially immediately following ingestion of the dosage form, a second group releases drug approximately 3-5 hours following ingestion, and an optional third group provides drug release after approximately 7-9 hours.
In a further alternative embodiment, the individual dosage units are compacted in a single tablet, and represent integral but discrete segments thereof (e.g., layers). For example, drug-containing particles or drug-containing beads can be compressed together into a single tablet using conventional tabletting means.
As will be appreciated by those skilled in the art and as described in the pertinent texts and literature, a number of methods are available for preparing drug-containing tablets or other dosage units which provide a variety of drug release profiles. Such methods include coating a drug or drug-containing composition, increasing the drug""s particle size, placing the drug within a matrix, and forming complexes of the drug with a suitable complexing agent.
The delayed release dosage units in the present capsules can be prepared, for example, by coating a drug or a drug-containing composition with a selected membrane coating material, typically although not necessarily a polymeric material. When a coating is used to provide delayed release dosage units, particularly preferred coating materials comprise bioerodible, gradually hydrolyzable and/or gradually water-soluble polymers. The xe2x80x9ccoating weight,xe2x80x9d or relative amount of coating material per dosage unit, generally dictates the time interval between ingestion and drug release.
Suitable membrane coating materials for effecting delayed release include, but are not limited to: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropylmethyl cellulose phthalate, cellulose ester-ether phthalate, hydroxypropylcellulose phthalate, alkali salts of cellulose acetate phthalate, alkaline earth salts of cellulose acetate phthalate, hydroxypropylmethyl cellulose hexahydrophthalate, cellulose acetate hexahydrophthalate, and carboxymethylcellulose sodium; acrylic acid polymers and copolymers preferably formed from acrylic acid, methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, e.g. copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate, with a terpolymer of ethyl acrylate, methyl methacrylate and trimethylammonioethyl methacrylate chloride (sold under the tradename Eudragit RS) particularly preferred; vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymers; and shellac, ammoniated shellac, shellac-acetyl alcohol, and shellac n-butyl stearate.
In some cases, it may be desirable for the third tablet or bead or particle fraction to provide for release of the active agent in the colon, in which case polymeric or other materials are used that enable drug release within the colon. These may be selected from the aforementioned list, or other materials may be used as will be known to those skilled in the art of pharmaceutical formulation and drug delivery. For example, hydrocolloid gums may be effective to provide for colonic delivery, e.g., guar gum, locust gum, bena gum, gum tragacanth, and karaya gum (see, e.g., U.S. Pat. No. 5,656,294 to Friend). Other materials suitable for effecting colonic drug delivery include polysaccharides, mucopolysaccharides, and related compounds, e.g., pectin, arabinogalactose, chitosan, chondroitin sulfate, dextran, galactomannan, and xylan.
Combinations of different coating materials may also be used to coat a single dosage unit.
To bring about the desired pulsatile release profile for a dosage form comprised of encapsulated tablets, the first tablet is provided with little or no coating material, the second tablet is provided with some degree of coating material, the coating weight of a third tablet is still higher, and so on. Analogously, for encapsulated dosage forms in which the drug-containing dosage units are beads or particles, a first fraction of beads or particles is provided with little or no coating material, a second fraction is provided with some degree of coating material, the coating weight of a third fraction is still higher, etc. For example, when the dosage form contains three tablets (or, analogously, three groups of drug-containing particles or beads), the first tablet, which releases drug substantially immediately, may have a total coating weight of less than about 10%, preferably less than about 8%, the second tablet may have a total coating weight in the range of approximately 10% to 30%, preferably 15% to 25%, and the third tablet, if present, may have a total coating weight in the range of approximately 15% to 65%, preferably 20% to 65%. The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for dosage units prepared with different quantities of various coating materials.
Alternatively, the delayed release dosage units, i.e., tablets or particles, may be formulated by dispersing the drug within a matrix of a suitable material such as an insoluble plastic, a hydrophilic polymer, or a fatty compound. The insoluble plastic matrices may be comprised of, for example, polyvinyl chloride or polyethylene. Hydrophilic polymers useful for providing a matrix for a delayed release dosage unit include, but are not limited to, those described above as suitable coating materials. Fatty compounds for use as a matrix material include, but are not limited to, waxes generally (e.g., carnauba wax) and glyceryl tristearate. Once the active ingredient is mixed with the matrix material, the mixture can be compressed into tablets or processed into individual drug-containing particles.
The individual dosage units may be provided with colored coatings, with a single color used to identify a tablet or bead or particle fraction having a corresponding delayed release profile. That is, for example, a blue coating may be used for the immediate release tablet or bead or particle fraction, a red coating may be used for the xe2x80x9cmediumxe2x80x9d release tablet or bead or particle fraction, and the like. In this way, errors during manufacture can be easily avoided. The color is introduced by incorporating a pharmaceutically acceptable colorant into the coating during coating preparation. The colorant may be either natural or synthetic. Natural colorants include pigments such as chlorophyll, anattenes, beta-carotene, alizarin, indigo, rutin, hesperidin, quercitin, carminic acid, and 6,6xe2x80x2-dibromoindigo. Synthetic colorants are dyes, including both acidic dyes and basic dyes, such as nitroso dyes, nitro dyes, azo dyes, oxazines, thiazines, pyrazolones, xanthenes, indigoids, anthraquinones, acridines, rosanilines, phthaleins, quinolines. e.g., a dye or pigment, during preparation of the coating solution.
For encapsulated tablets, the weight of each individual tablet in the capsule is typically in the range of about 10 mg to 150 mg, preferably in the range of about 25 mg to about 100 mg, and most preferably is in the range of about 40 mg to 80 mg. The individual tablets are prepared using conventional means. A preferred method for forming tablets herein is by direct compression of a powdered, crystalline or granular drug-containing composition, alone or in combination with diluents, binders, lubricants, disintegrants, colorants or the like. As an alternative to direct compression, compressed tablets can be prepared using wet-granulation or dry-granulation processes. Tablets may also be molded rather than compressed, starting with a moist material containing a suitable water-soluble lubricant. Preferred tablets herein are manufactured using compression rather than molding, however. Drug-containing particles or beads are also prepared using conventional means, typically from a fluid dispersion.
Conventional coating procedures and equipment may then be used to coat the dosage units, i.e., the drug-containing tablets, beads or particles. For example, a delayed release coating composition may be applied using a coating pan, an airless spray technique, fluidized bed coating equipment, or the like. For detailed information concerning materials, equipment and processes for preparing tablets, beads, drug particles, and delayed release dosage forms, reference may be had to Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and to Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th Ed. (Media, PA: Williams and Wilkins, 1995).
Optional components present in the individual drug-containing dosage units include, but are not limited to, diluents, binders, lubricants, disintegrants, stabilizers, surfactants, coloring agents, and the like. Diluents, also termed xe2x80x9cfillers,xe2x80x9d are typically necessary to increase the bulk of a tablet so that a practical size is provided for compression. Suitable diluents include, for example, dicalcium phosphate dihydrate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, hydrolyzed starches, silicon dioxide, titanium oxide, alumina, talc, microcrystalline cellulose, and powdered sugar. Binders are used to impart cohesive qualities to a tablet formulation, and thus ensure that a tablet remains intact after compression. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums, e.g., acacia, tragacanth, sodium alginate, polyvinylpyrrolidone, celluloses, and Veegum, and synthetic polymers such as polymethacrylates and polyvinylpyrrolidone. Lubricants are used to facilitate tablet manufacture; examples of suitable lubricants include, for example, magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, and polyethylene glycol, and are preferably present at no more than approximately 1 wt.% relative to tablet weight. Disintegrants are used to facilitate tablet disintegration or xe2x80x9cbreakupxe2x80x9d after administration, and are generally starches, clays, celluloses, algins, gums or crosslinked polymers. Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions. Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents, with anionic surfactants preferred. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions, associated with cations such as sodium, potassium and ammonium ions. Particularly preferred surfactants include, but are not limited to: long alkyl chain sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylhexyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. If desired, the tablets may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
As noted earlier herein, the individual drug tablets, beads or particles are, in one embodiment, contained within a closed capsule. The capsule material may be either hard or soft, and as will be appreciated by those skilled in the art of pharmaceutical science, typically comprises a tasteless, easily administered and water soluble compound such as gelatin, starch or cellulose. A preferred capsule material is gelatin. The capsules are preferably sealed, such as with gelatin bands or the like. See, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Edition (Easton, PA: Mack Publishing Co., 1995), which describes materials and methods for preparing encapsulated pharmaceuticals designed to dissolve shortly after ingestion.
The novel dosage forms are used to administer methylphenidate in a pulsatile release manner. As noted earlier herein, the drug is administered as a racemic mixture of the d-threo and l-threo enantiomers. For administration of racemic methylphenidate, a dosage form of the invention comprises a total of approximately 2 mg to 100 mg methylphenidate, preferably 2 mg to 50 mg methylphenidate, divided among the individual dosage units. Optimally, the first and second dosage units each contain approximately 2 mg to 20 mg methylphenidate, and preferably, the first and second methylphenidate doses are approximately the same. The third tablet should contain a lower dose of methylphenidate, preferably about half the dose in the first tablet, to avoid sleep disruption. Also, if an additional CNS stimulant such as d-amphetamine is incorporated into the dosage form, it will be included in the first, immediate release dosage unit, will optionally be present in the second dosage unit, (and if present, at a lower dose than in the first dosage unit), and will not be included in the third dosage unit.
It may be desirable to include one or more additional active agents in the dosage forms herein. These active agents may potentiate certain effects of methylphenidate, or vice versa. The additional active agent or agents may be combined with methylphenidate in a single dosage unit within the dosage form, or one or more dosage units within the dosage form may comprise the additional active agent without any methylphenidate. In the former case, the various active agents may be present as an admixture in a tablet, or the agents may be physically segregated as in a bilayer tablet, a tablet having two or more active agent-containing coatings, or the like.
Preferred additional active agents, i.e., active agents for co-administration with methylphenidate, are CNS stimulants (including analeptic agents and psychostimulants), antidepressant drugs and antianxiety agents. Particularly preferred are CNS stimulants including, but not limited to: amphetamine (racemic), d-amphetamine, amphetamine and d-amphetamine phosphate, amphetamine and d-amphetamine sulfate, amphetamine and d-amphetamine hydrochloride, amphetamine and d-amphetamine saccharate, and amphetamine and d-amphetamine aspartate, amphetaminil, bemegride, benzphetamine, benzphetamine hydrochloride, brucine, chlorphentermine, clofenciclan, clortermine, deanol acetamidobenzoate, demanyl phosphate, dexoxadrol, diethpropion, doxapram hydrochloride, N-ethylamphetamine, ethamivan, etifelmin, etryptamine, fencamfamine, fenethylline, fenosolone, fenfluramine, flurothyl, hexacyclonate sodium, homocamfin, mazindol, megexamide, methamphetamine, nicotinic agonists, nikethamide, pemoline, pentylenetetrazole, phenidimetrazine, phendimetrazine tartrate, phenmetrazine, phenmetrazine hydrochloride, phentermine, picrotoxin, pipradrol, pipradrol hydrochloride, prolintane, pyrovalerone, racephedrine, racephedrine hydrochloride, and tetrahydrobenzothienopyridines. Pemoline, amphetamine, d-amphetamine and salts thereof are particularly preferred additional active agents.
Antidepressant drugs include, for example: tricyclic antidepressants such as imipramine, amitryptyline, amoxapine, clomipramine, desipramine, doxepin, imipramine, maprotiline, nortriptyline, protriptyline and trimipramine; monoamine oxidase inhibitors (MAOIs) such as isocarboxazid, phenelzine, selegiline and tranylcypromine; selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, fluvoxamine, paroxetine, sertraline, venlaxafine, and the like; and other antidepressants including buproprion, nefazodone and trazodone.
Examples of antianxiety agents include, but are not limited to, benzodiazepines such as alprazolam, chlordiazepoxide, clonazepam, clorazepate, diazepam, estazolam, flurazepam, halazepam, lorazepam, midazolam, oxazepam, prazepam, quazepam, temazepam and triazolam; carbamates such as meprobamate and ethinamate; chloral hydrate and related drugs; piperidine-diones such as glutethimide and methyprylon; alcohols such as ethchlorvynol; antihistaminics such as diphenhydramine and hydroxyzine; methaqualone; and paraldehyde.
Each of the active agents in the individual tablets may be in the form of a pharmaceutically acceptable salt, ester, amide, prodrug or other derivative or analog, including active agents modified by appending one or more appropriate functionalities to enhance selected biological properties. Such modifications are known in the art and/or are described in the pertinent texts and literature.
Salts of the active agents used in conjunction with the present dosage forms may be obtained commercially or can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992). Suitable acids for preparing acid addition salts may be weak acids, medium acids, or strong acids, and include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, aspartic acid, saccharic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Preparation of basic salts of acid moieties which may be present (e.g., carboxylic acid groups) are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, magnesium hydroxide, trimethylamine, or the like. Preparation of esters involves functionalization of hydroxyl and/or carboxyl groups which may be present. These esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties which are derived from carboxylic acids of the formula RCOOH where R is alkyl, and preferably is lower alkyl. Pharmaceutically acceptable esters may be prepared using methods known to those skilled in the art and/or described in the pertinent literature. Amides, prodrugs, and other analogs and derivatives can be readily prepared as well, using conventional means.
Utility
The novel drug dosage forms are to be administered orally to a mammalian individual and can be used to administer methylphenidate to treat or prevent a variety of disorders, conditions and diseases. In accordance with the present invention, administration of methylphenidate may be carried out in order to treat any disorder, condition or disease for which methylphenidate is generally indicated. Such disorders, conditions and diseases include, for example, ADD, ADHD, narcolepsy, and acute depression; methylphenidate may also be used in the treatment of individuals suffering from cognitive decline associated with AIDS or AIDS-related conditions, and for mood elevation in terminally ill patients suffering from a disease such as cancer.
For administration of racemic methylphenidate, the typical daily dose is in the range of approximately 5 mg to 100 mg, preferably 5 mg to 60 mg, although the exact dosage regimen will depend on a number of factors, including age, the general condition of the patient, the particular condition or disorder being treated, the severity of the patient""s condition or disorder, and the like.
It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the description above as well as the examples which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Pharmaceutical dosage form for pulsatile delivery of methylphenidate


A method to protect corn against feeding damage by one or more pests includes the treatment of corn seed having a transgenic event that is targeted against at least one of the pests with a pesticide in an amount that is effective against the same or another of the one or more pests. Seeds having such protection are also disclosed.

(1) Field of the Invention
The present invention relates generally to the control of pests that cause damage to corn plants by their feeding activities, and more particularly to the control of such corn plant pests by the combination of a corn seed having a transgenic event and the treatment of such seed with a pesticide prior to planting the seed.
(2) Description of the Related Art
Insects and related arthropods annually destroy an estimated 15% of agricultural crops in the United States and even more than that in developing countries. In addition, competing weeds and parasitic and saprophytic plants account for even more potential yield losses.
Some of this damage occurs in the soil when plant pathogens, insects and other such soil borne pests attack the seed after planting. In the production of corn, much of the rest of the damage is caused by rootwormsxe2x80x94insect pests that feed upon or otherwise damage the plant roots; and by cutworms, European corn borers, and other pests that feed upon or damage the above ground parts of the plant. General descriptions of the type and mechanisms of attack of pests on agricultural crops are provided by, for example, Metcalf, in Destructive and Useful Insects, (1962); and Agrios, in Plant Pathology, 3rd Ed., Academic Press (1988).
Corn is the most important grain crop in the Midwestern United States. Among the most serious insect pests of corn in this region is the larval form of three species of Diabrotica beetles. These include the Western corn rootworm, Diabrotica vergifera vergifera LeConte, the Northern corn rootworm, Diabrotica berberi Smith and Diabrotica berberi Lawrence, and the Southern corn rootworm, Diabrotica undecimpunctata howardi Barber. In fact, more insecticide is used for the control of corn rootworm than for any other pest of corn, and the total acreage treated is greater than for any other pest in the United States.
Corn rootworms (CRW) overwinter in the egg stage in fields where corn was grown the previous season. The eggs hatch from late May through June. If a corn crop is not followed by another corn crop in the subsequent year, the larvae will die. Accordingly, the impact of corn rootworm is felt most directly in areas where corn is systematically followed by corn, as is typical in many areas of the Midwestern United States.
After hatching, the larvae pass through three larval stages or instars, during which they feed on the corn root system. About three weeks is required for completion of the larval stage. Damage to the corn root system caused by the feeding of larvae is the major cause of harvest losses in corn due to corn rootworm. Corn plants that fall over and lodge in the field after weakening or destruction of a major part of the root system are the cause of a major portion of this loss, since this lodged corn cannot be harvested by conventional mechanized machinery and is left in the field.
Following completion of larval development, the larvae transform into immobile pupae, and thence into the adult beetles that emerge from the soil throughout the summer, with the period of emergence depending upon the growing location. After emergence, the adult beetles feed for about two weeks before the females start laying eggs. Initially, the adults feed predominantly in the same field from which they emerged, but later will migrate to other fields. Peak adult activity normally occurs in the U.S. Corn Belt during late July or early August in fields planted to continuous corn, but activity may peak later in first year or late maturing cornfields. Rootworm beetles begin depositing eggs in cornfields approximately two weeks after they emerge. (For more information, see, e.g., Corn Rootworms, Field Crops Pest Management Circular #16, Ohio Pest Management and Survey Program, The Ohio State University, Extension Division, Columbus, Ohio.; available online at www.ag.ohio-state.edu/xcx9cohioline/icm-fact/fc-16.html, Sep. 13, 2000; and McGahen et al., Corn Insect Control: Corn Rootworm, PENpages number 08801502, Factsheet available from Pennsylvania State University, State College, Pa., 1989).
In present conventional agricultural practice, in cases where corn follows corn, it is normal for an insecticide to be applied to protect the corn root system from severe feeding by rootworm larvae. Conventional practice is to treat for the adult beetles or to treat for the larvae. Examples of conventional treatment formulations for adult beetles include the application of carbaryl insecticides (e.g., SEVIN(copyright) 80S at 1.0-2.0 lbs active/acre); fenvalerate or esfenvalerate (e.g., PYDRIN(copyright) 2.4EC at 0.1 to 0.2 lbs active/acre, or ASANA(copyright) 0.66EC at 0.03 to 0.05 lbs active/acre); malathion (57% E at 0.9 lbs active/acre); permethrin (e.g., AMBUSH(copyright) 2.OEC at 0.1 to 0.2 lbs active/acre, or POUNCE(copyright) 3.2EC at 0.1 to 0.2 lbs active ingredient/acre); or PENNCAP-M(copyright) at 0.25-0.5 lbs active/acre.
To treat for CRW larvae, conventional practice is to apply a soil insecticide either at or after planting, but preferably as close to egg hatching as possible. Conventional treatments include carbofuran insecticides (e.g., FURADAN(copyright) 15G at 8 oz/1000 ft of row); chloropyrifos (e.g., LORSBAN(copyright) 15G at 8 oz/1000 ft of row); fonophos (e.g., DYFONATE(copyright) 20G at 4.5 to 6.0 oz/1000 ft of row); phorate (e.g., THIMET(copyright) 20G at 6 oz/1000 ft of row); terbufos (e.g., COUNTER(copyright) 15G at 8 oz/1000 ft of row); or tefluthrin (e.g., FORCE(copyright) 3G at 4 to 5 oz/1000 ft of row).
Many of the chemical pesticides listed above are known to be harmful to humans and to animals in general. The environmental harm that these pesticides cause is often exacerbated due to the practice of applying the pesticides by foliar spraying or direct application to the surface of the soil. Wind-drift, leaching, and runoff can cause the migration of a large fraction of the pesticide out of the desired zone of activity and into surface waters and direct contact with birds, animals and humans.
Because of concern about the impact of chemical pesticides on public health and the health of the environment, significant efforts have been made to find ways to reduce the amount of chemical pesticides that are used. Recently, much of this effort has focused on the development of transgenic crops that are engineered to express insect toxicants derived from microorganisms. For example, U.S. Pat. No. 5,877,012 to Estruch et al. discloses the cloning and expression of proteins from such organisms as Bacillus, Pseudomonas, Clavibacter and Rhizobium into plants to obtain transgenic plants with resistance to such pests as black cutworms, armyworms, several borers and other insect pests. Publication WO/EP97/07089 by Privalle et al. teaches the transformation of monocotyledons, such as corn, with a recombinant DNA sequence encoding peroxidase for the protection of the plant from feeding by corn borers, earworms and cutworms. Jansens et al., in Crop Sci., 37(5):1616-1624 (1997), reported the production of transgenic corn containing a gene encoding a crystalline protein from Bacillus thuringiensis (Bt) that controlled both generations of the European corn borer. U.S. Pat. Nos. 5,625,136 and 5,859,336 to Koziel et al. reported that the transformation of corn with a gene from B. thuringiensis that encoded for delta-endotoxins provided the transgenic corn with improved resistance to European corn borer. A comprehensive report of field trials of transgenic corn that expresses an insecticidal protein from B. thuringiensis has been provided by Armstrong et al,, in Crop Science, 35(2):550-557 (1995).
It was known that wild-type Bt xcex4-endotoxins had low activity against coleopteran insects, and Kreig et al., in 1983, reported the first isolation of a coleopteran-toxic B. thuringiensis strain. (See U.S. Pat. No. 4,766,203). U.S. Pat. Nos. 4,797,279 and 4,910,016, also disclosed wild-type and hybrid B. thuringiensis strains that produced proteins having some coleopteran activity. More recently, however, more precise genetic engineering methods have shown promise in developing modified B. thuringiensis proteins that have significantly higher levels of corn rootworm activity than those produced by wild-type parents. (See, e.g., WO 99/31248 to Ecogen, Inc. and Monsanto Company).
However, it is not known at present whether any transgenic event alone will be sufficiently effective to protect corn from damage by corn rootworm in heavily infested fields that are dedicated to serial corn. In fact, the total control of corn rootworm damage by any one transgenic event may not be desirable in the long term, because of the potential for the development of resistant strains of the target pest.
Another alternative to the conventional forms of pesticide application is the treatment of plant seeds with pesticides. The use of fungicides to protect seeds from attack after planting, and the use of low levels of insecticides for the protection of, for example, corn seed from wireworm, has been used for some time. Seed treatment with pesticides has the advantages providing for the protection of the seeds, while minimizing the amount of pesticide that was required and limiting the amount of contact with the pesticide and the number of different field applications that were necessary.
Other examples of the control of pests by applying insecticides directly to plant seed are provided in, for example, U.S. Pat. No. 5,696,144, which discloses that the European corn borer caused less feeding damage to corn plants grown from seed treated with a 1-arylpyrazole compound at a rate of 500 g per quintal of seed than control plants grown from untreated seed. In addition, U.S. Pat. No. 5,876,739 to Turnblad et al. (and its parent, U.S. Pat. No. 5,849,320) disclose a method for controlling soil-borne insects which involves treating seeds with a coating containing one or more polymeric binders and an insecticide. This reference provides a list of insecticides that it identifies as candidates for use in this coating and also names a number of potential target insects. However, while the U.S. Pat. No. 5,876,739 states that treating corn seed with a coating containing a particular insecticide protects corn roots from damage by the corn rootworm, it does not indicate or otherwise suggest that such treatment could be used with seed having a transgenic event.
The treatment of seed having a transgenic event with nitroimino- or nitroguanidino-compound pesticides has been mentioned (See, e.g., WO 99/35913), however, no guidance has been found as to the potential utility or efficacy of such treatments, or the details of how such treatments might be effectedxe2x80x94such as the amounts of active ingredient that would be necessary per unit amount of seedxe2x80x94and no examples that would give reason to believe that the proposed treatments would actually provide suitable protection.
Therefore, although recent developments in genetic engineering of plants have improved the ability to protect plants from pests without using chemical pesticides, and while such techniques as the treatment of seeds with pesticides have reducing the harmful effects of pesticides on the environment, numerous problems remain that limit the successful application of these methods under actual field conditions. Accordingly, it would be useful to provide an improved method for the protection of plants, especially corn plants, from feeding damage by pests. It would be particularly useful if such method would reduce the required application rate of conventional chemical pesticides, and also if it would limit the number of separate field operations that were required for crop planting and cultivation.
Briefly, therefore, the present invention is directed to a novel method for protecting a transgenic corn plant against feeding damage by one or more pests, the method comprising providing a seed for the transgenic corn plant which seed comprises a transgenic event having activity against at least one of the one or more pests; and treating the seed with an effective amount of a pesticide.
The present invention is also directed to a novel seed of a transgenic corn plant that provides increased resistance to the resulting corn plant against feeding damage by one or more pests, comprising a transgenic event having activity against at least one of the one or more pests, which seed has been treated with an effective amount of a pesticide.
The present invention is also directed to a novel transgenic corn seed that has been treated by the method of the present invention.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of an improved method for the protection of plants, especially corn plants, from feeding damage by pests; the provision of such a method that would reduce the required application rate of conventional chemical pesticides; and also the provision of such a method that would limit the number of separate field operations that were required for crop planting and cultivation.
In accordance with the present invention, it has been discovered that corn plants can be protected against feeding damage by one or more pests by a method that includes providing a corn seed having a transgenic event that has activity against at least one of the pests and then treating the transgenic corn seed with an effective amount of a pesticide. In preferred embodiments of this invention, it has been found that the combination of a transgenic event having activity against corn rootworm and treatment of the seed with certain pesticides provides unexpectedly synergistic advantages to seeds having such treatment, including unexpectedly superior efficacy for protection against damage to the resulting corn plant by corn rootworm. In particular, it was found that corn seeds having a transgenic event that caused the production of CryBb.11231 endotoxin in combination with the treatment of such seeds with imidacloprid was unexpectedly superior to either the transgenic event alone, or to treatment with imidacloprid alone, in protecting resulting corn plants against more severe levels of damage by corn rootwormxe2x80x94levels of damage that are known to reduce corn yield.
Corn plants and seeds that have been engineered to include exogenous genes derived from Bacillus thuringiensis that encode for the expression of Cry3 xcex4-endotoxins having activity against Coleopteran pests are known, as are methods for the treatment of seeds (even some transgenic seeds) with pesticides. However, it had not been realized until the present invention that certain effective amounts of certain pesticides could be used to treat corn seeds having such Cry3 events, with the result that the combination would be effective, and preferably unexpectedly superior, in increasing the efficacy of both the pesticide and the transgenic event, and would provide the additional advantages of increasing the ability to match pesticidal activity against pest pressure, decreasing cost of treatment and/or application, increasing safety of seed handling, and decreasing environmental impact of either or both the event and the pesticide.
In particular, it has been found in preferred embodiments that the treatment of a transgenic corn seeds that are capable of expressing certain modified Cry3Bb proteins with from about 100 gm to about 400 gm of certain pesticides per 100 kg of seed provided unexpectedly superior protection against corn rootworm. In addition, it is believed that such combinations are also effective to protect the emergent corn plants against damage by black cutworm. The seeds of the present invention are also believed to have the property of decreasing the cost of pesticide use, because less of the pesticide can be used to obtain a required amount of protection than if the innovative method is not used. Moreover, because less pesticide is used and because it is applied prior to planting and without a separate field application, it is believed that the subject method is therefore safer to the operator and to the environment, and is potentially less expensive than conventional methods.
When it is said that some effects are xe2x80x9csynergisticxe2x80x9d, it is meant to include the synergistic effects of the combination on the pesticidal activity (or efficacy) of the combination of the transgenic event and the pesticide. However, it is not intended that such synergistic effects be limited to the pesticidal activity, but that they should also include such unexpected advantages as increased scope of activity, advantageous activity profile as related to type and amount of damage reduction, decreased cost of pesticide and application, decreased pesticide distribution in the environment, decreased pesticide exposure of personnel who produce, handle and plant corn seeds, and other advantages known to those skilled in the art.
The present invention also provides an advantage of increasing the ability to match pesticidal activity against pest pressure. This refers to the ability to design the combination of the transgenic event and the pesticide treatment so that the seed or the resulting plant is provided with effective pesticidal activity during the period when feeding pressure from the target pest on the seed or plant reaches its maximum. By way of example, when a pesticide such as imidacloprid or terfluthrin is applied to a corn seed having a corn rootworm transgenic event, the pesticide can be applied in a coating designed to provide controlled release of the pesticide. The release rate can be selected so that the pesticide provides protection against such other pests as, for example, black cutworm, at the post emergence stage of corn, while the transgenic event provides corn rootworm protection at a later stage of plant developmentxe2x80x94when such protection is needed.
As used herein, the terms xe2x80x9cpesticidal effectxe2x80x9d and xe2x80x9cpesticidal activityxe2x80x9d, or xe2x80x9cactivityxe2x80x9d refer to a toxic effect against a pest. The terms xe2x80x9cactivity against (one or more) pestsxe2x80x9d, also have the same meaning. When it is said that a seed or plant is xe2x80x9cprotected against feeding damage by one or more pestsxe2x80x9d, it is meant that such seed or plant possesses a feature having direct or indirect action on one or more pests that results in reduced feeding damage by such pest or pests on the seeds, roots, shoots and foliage of plants having such feature as compared to the feeding damage caused under the same conditions to plants not having such feature. Such direct or indirect actions include inducing death of the pest, repelling the pest from the plant seeds, roots, shoots and/or foliage, inhibiting feeding of the pest on, or the laying of its eggs on, the plant seeds, roots, shoots and/or foliage, and inhibiting or preventing reproduction of the pest.
The term xe2x80x9cinsecticidal activityxe2x80x9d has the same meaning as pesticidal activity, except it is limited to those instances where the pest is an insect. Except where specifically noted, when the term xe2x80x9cpesticidexe2x80x9d is used herein, that term refers to a chemical pesticide that is supplied externally to the seed, and it is not meant to include active agents that are produced by the particular seed or the plant that grows from the particular seed. However, the terms xe2x80x9cpesticidal activityxe2x80x9d and xe2x80x9cinsecticidal activityxe2x80x9d can be used with reference to the activity of either, or both, an externally supplied pesticide and/or an agent that is produced by the seed or the plant.
One feature of the present invention is a seed of a transgenic corn plant. As used herein, the terms xe2x80x9ctransgenic corn plantxe2x80x9d mean a corn plant or progeny thereof derived from a transformed corn plant cell or protoplast, wherein the plant DNA contains an introduced exogenous DNA molecule not originally present in a native, non-transgenic plant of the same strain.
The transgenic corn seed is one that contains an exogenous gene that encodes a pesticidal protein. Pesticidal proteins of this type are described by Schnepf et al., in Microbiology and Molecular Biology Reviews, 62:775-806 (1998), and by ffrench-Constant and Bowen, in CMSL Cell. Mol. Life Sci., 57:828-833 (2000). In one application of the invention, the pesticidal protein is an insecticidal protein.
It is preferred that the seed contains an exogenous gene derived from a strain of Bacillus thuringiensis, and in particular, it is preferred that the exogenous gene is one that encodes an insecticidal xcex4-endotoxin derived from B. thuringiensis. Such xcex4-endotoxins are described in WO 99/31248 and U.S. Pat. No. 6,063,597, and include the Cry3 toxins. Nucleic acid segments that encode modified B. thuringiensis coleopteran-toxic crystal proteins that are useful in the present invention are described in U.S. Pat. No. 6,060,594, and insect resistant transgenic plants that include nucleic acid sequences that encode such insecticidal proteins are discussed in U.S. Pat. No. 6,023,013. It is preferred that the xcex4-endotoxins of the present invention include the Cry3B proteins, and even more preferred that the xcex4-endotoxins include the coleopteran-active Cry3Bb proteins. The nomenclature of the B. thuringiensis insecticidal crystal proteins was set forth by Hofte and Whitely, Microbiol. Rev.,53:242-255, 1989. This nomenclature has been revised, and the revised nomenclature can be found at http://epunix.biols.susx.ac.uk/Home/Neil-Crickmore/Bt/index.html. The revised nomenclature will be used herein to describe transgenic event features and the xcex4-endotoxin proteins encoded by the transgenic event.
When the terms xe2x80x9ctransgenic eventxe2x80x9d are used herein, such terms are meant to refer to the genetically engineered DNA that is described above, but also to include the protein(s) that are encoded by the modified gene. A transgenic event in a corn seed, or corn plant, therefore, includes the ability to express a protein. When it is said that a xe2x80x9ctransgenic event has activity against a pestxe2x80x9d, it is to be understood that it is the protein that is encoded by the gene that actually has such activity when the protein is expressed and brought into contact with the pest.
WO 99/31248 and U.S. Pat. No. 6,063,597 describe methods for genetically engineering B. thuringiensis xcex4-endotoxin genes so that modified xcex4-endotoxins can be expressed. The modified xcex4-endotoxins differ from the wild-type proteins by having specific amino acid substitutions, additions or deletions as compared with the proteins produced by the wild-type organism. Such modified xcex4-endotoxins are identified herein by the use of an asterisk (*), or by reference to a specific protein by its identifying number. Thus, a genetically modified Cry3 xcex4-endotoxin would be expressed as Cry3*, examples of which include, without limitation: Cry3Bb.11230, Cry3Bb.11231, Cry3Bb. 11232, Cry3Bb. 11233, Cry3Bb. 11234, Cry3Bb.11235, Cry3Bb. 11236, Cry3Bb. 11237, Cry3Bb. 11238, Cry3Bb.11239, Cry3Bb.11241, Cry3Bb.11242, and Cry3Bb.11098.
Some of the modified xcex4-endotoxins that were described in WO 99/31248 and in U.S. Pat. No. 6,063,597 were found to have enhanced activity against coleopteran insects, and in particular against Diabrotica spp., including corn rootworm. As used herein, the terms xe2x80x9cenhanced activityxe2x80x9d refer to the increased insecticidal activity of a modified toxin as compared with the activity of the same toxin without the amino acid modifications when both are tested under the same conditions. In particular, it was found that Cry3* xcex4-endotoxins had enhanced activity against corn rootworm, and are therefore preferred for use in the present invention. More preferred are Cry3B* xcex4-endotoxins, and even more preferred are Cry3Bb* xcex4-endotoxins. Even more preferred transgenic events are those that comprise the ability to express the modified xcex4-endotoxins that are listed in the following table. Also shown in the table are strains of transgenic B. thuringiensis that include genes for expression of the respective novel endotoxins, and the date and accession number of their deposit with the Agricultural
Research Service Collection (NRRL) at 1815 N. University Street, Peoria, Ill. 91904.
It has also been found that a preferred use of the present invention is for reducing pest feeding damage when used in combination with seeds having transgenic events that have certain levels of effectiveness against such pest. To illustrate which levels of effectiveness are preferred, the following example will use the Iowa Root Rating Method (Hills and Peters, J. Econ. Entomol., 64:764-765, 1971), which measures corn rootworm feeding damage to corn roots on a 1-6 scale. In the rating, 1=no damage or only a few minor feeding scars; 2=feeding scars evident but no roots eaten off to within 1xc2xd inch of the plant; 3=several roots eaten off to within 1xc2xd inch of the plant, but never the equivalent of an entire node of roots is destroyed; 4=one root node completely destroyed; 5=two root nodes completely destroyed; and 6=three or more root nodes destroyed. A destroyed root is defined as a root that has been pruned to within 1xc2xd inch of the base. Pruned roots do not have to originate from a single node, but all pruned roots must equal the equivalent of a full node to count as a destroyed node.
As used herein, a transgenic event is within the preferred range of effectiveness level against a target pest if that event reduces feeding damage by that pest by a certain amount as compared with the same crop without the transgenic event, but does not prevent substantially all damage by the target pest. For example, if 10% of transgenic corn suffered corn rootworm damage of 4 or higher on the Iowa 1-6 Scale, while 80% of non-transgenic corn suffered damage of 4 or higher, then it could be said that the damage to the transgenic corn was (10/80)xc3x97100=12.5% of that of the non-transgenic corn. For the purposes of the present invention, it will be understood that a transgenic event in corn is within the preferred range of effectiveness level if corn having such event suffers from about 5% to about 50% of the damage suffered by non-transgenic corn due to the same pest under the same conditions. It is more preferred that corn having such transgenic event suffers from about 10% to about 40% of the damage suffered by non-transgenic corn by the same pest under the same conditions, even more preferred is damage of from about 15% to about 30%, and yet more preferred is damage of from about 20% to about 30% of the damage suffered by non-transgenic corn by the same pest under the same conditions. As used herein, when the term xe2x80x9caboutxe2x80x9d is used to describe the degree of damage to corn, it is to be understood that the degree of damage can be above or below the limits described by as much as 1% or 2% and still be considered to be within the ranges described. By way of example, a level of 4.5% damage would be regarded as being xe2x80x9cabout 5%xe2x80x9d.
Without wishing to be bound to this or any other theory, it is believed that the pesticidal seed treatment can provide significant advantages when combined with a transgenic event that provides protection that is within the preferred effectiveness range against a target pest. In addition, it is believed that there are situations that are well known to those having skill in the art, where it is advantageous to have such transgenic events within the preferred range of effectiveness.
The present invention also includes seeds and plants having more that one transgenic event. Such combinations are referred to as xe2x80x9cstackedxe2x80x9d transgenic events. These stacked transgenic events can be events that are directed at the same target pest, or they can be directed at different target pests. In one preferred method, a seed having the ability to express a Cry 3 protein also has the ability to express at least one other insecticidal protein that is different from a Cry 3 protein.
In another preferred method, the seed having the ability to express a Cry 3 protein also has a transgenic event that provides herbicide tolerance. It is more preferred that the transgenic event that provides herbicide tolerance is an event that provides resistance to glyphosate, N-(phosphonomethyl) glycine, including the isopropylamine salt form of such herbicide, even more preferred is the transgenic event that is effective to provide the herbicide resistance of ROUNDUP READY(copyright) plants and seeds available from Monsanto Co., St. Louis, Mo.
In the present method, a corn seed having a transgenic event is treated with a pesticide.
Pesticides suitable for use in the invention include pyrethrins and synthetic pyrethroids; oxadizine derivatives; chloronicotinyls; nitroguanidine derivatives; triazoles; organophosphates; pyrrols; pyrazoles; phenyl pyrazoles; diacylhydrazines; biological/fermentation products; and carbamates. Further information about pesticides within these categories can be found, for example, in The Pesticide Manual, 11th Ed., C. D. S. Tomlin, Ed., British Crop Protection Council, Farnham, Surry, UK (1997).
Pyrethroids that are useful in the present composition include pyrethrins and synthetic pyrethroids. The pyrethrins that are preferred for use in the present method include, without limitation, 2-allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of 2,2-dimethyl-3-(2methyl propenyl)-cyclopropane carboxylic acid, and/or (2-methyl-1-propenyl) -2-methoxy-4-oxo-3-(2 propenyl)-2-cyclopenten-1-yl ester and mixtures of cis and trans isomers thereof (Chemical Abstracts Service Registry Number (xe2x80x9cCAS RNxe2x80x9d) 8003-34-7).
Synthetic pyrethroids that are preferred for use in the present invention include (s)-cyano(3-phenoxyphenyl)methyl 4-chloro alpha (1-methylethyl)benzeneacetate (fenvalerate, CAS RN 51630-58-1), (S)-cyano (3-phenoxyphenyl) methyl (S)-4-chloro-alpha-(1-methylethyl) benzeneacetate (esfenvalerate, CAS RN 66230-04-4), (3-phenoxyphenyl)-methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylcyclopropanecarboxylate (permethrin, CAS RN 52645-53-1), (xc2x1) alpha-cyano-(3-phenoxyphenyl) methyl(+)-cis,trans-3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropane carboxylate (cypermethrin, CAS RN 52315-07-8), (beta-cypermethrin, CAS RN 65731-84-2), (theta cypermethrin, CAS RN 71697-59-1), S-cyano (3-phenoxyphenyl) methyl (xc2x1) cis/trans 3-(2,2-dichloroethenyl) 2,2 dimethylcyclopropane carboxylate (zeta-cypermethrin, CAS RN 52315-07-8), (s)-alpha-cyano-3-phenoxybenzyl (IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate (deltamethrin, CAS RN 52918-63-5), alpha-cyano-3-phenoxybenzyl 2,2,3,3,-tetramethyl cyclopropoanecarboxylate (fenpropathrin, CAS RN 64257-84-7), (RS)-alpha-cyano-3-phenoxybenzyl(R)-2-[2-chloro-4-(trifluoromethyl)anilino]-3-methylbutanoate (tau-fluvalinate, CAS RN 102851-06-9), (2,3,5,6-tetrafluoro-4-methylphenyl)-methyl-(1 alpha, 3 alpha)-(Z)-(xc2x1)-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate (tefluthrin, CAS RN 79538-32-2), (xc2x1)-cyano (3-phenoxyphenyl) methyl (xc2x1)-4-(difluoromethoxy)-alpha-(1-methyl ethyl) benzeneacetate (flucythrinate, CAS RN 70124-77-5), cyano(4-fluoro-3-phenoxyphenyl)methyl 3-[2-chloro-2-(4-chlorophenyl)ethenyl]-2,2-dimethylcyclopropanecarboxylate (flumethrin, CAS RN 69770-45-2), cyano(4-fluoro-3-phenoxyphenyl) methyl 3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanedarboxylate (cyfluthrin, CAS RN 68359-37-5), (beta cyfluthrin, CAS RN 68359-37-5), (transfluthrin, CAS RN 118712-89-3), (S)-alpha-cyano-3-phenoxybenzyl(Z)-(IR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethyl-ethoxycarbonyl)vinyl]cyclopropane carboxylate (acrinathrin, CAS RN 101007-06-1), (IR cis) S and (IS cis) R enantiomer isomer pair of alpha-cyano-3-phenoxybenzyl-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (alpha-cypermethrin, CAS RN 67375-30-8), [IR,3S)3(1xe2x80x2RS)(1xe2x80x2,2xe2x80x2,2xe2x80x2,2xe2x80x2-tetrabromoethyl)]-2,2-dimethyl cyclopropanecarboxylic acid (s)-alpha-cyano-3-phenoxybenzyl ester (tralomethrin, CAS RN 66841-25-6), cyano-(3-phenoxyphenyl) methyl 2,2-dichloro-1-(4-ethoxyphenyl)cyclopropane carboxylate (cycloprothrin, CAS RN 63935-38-6), [1xcex1,3xcex1(Z)]-(xc2x1)-cyano-(3-phenoxyphenyl)methyl 3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-cimethylcyclopropanecarboxylate (cyhalothrin, CAS RN 68085-85-8), [1 alpha (s), 3 alpha(z)]-cyano(3-phenoxyphenyl) methyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropane carboxylate (lambda cyhalothrin, CAS RN 91465-08-6), (2-methyl [1,1xe2x80x2-biphenyl]-3-yl) methyl 3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-cyclopropanecarboxylate (bifenthrin, CAS RN 82657-04-3), 5-1-benzyl-3-furylmethyl-d-cis(1R,3S,E)2,2-dimethyl-3-(2-oxo,-2,2,4,5 tetrahydro thiophenylidenemethyl)cyclopropane carboxylate (kadethrin, RU15525, CAS RN 58769-20-3), [5-(phenyl methyl)-3-furanyl]-3-furanyl 2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropane carboxylate (resmethrin, CAS RN 10453-86-8), (1 R-trans)-[5-(phenylmethyl)-3-furanyl]methyl 2,2-dimethyl-3-(2-methyl-1 -propenyl)cyclopropanecarboxylate (bioresmethrin, CAS RN 28434-01-7), 3,4,5,6-tetra hydro-phthalimidomethyl-(IRS)-cis-trans-chrysanthemate (tetramethrin, CAS RN 7696-12-0), 3-phenoxybenzyl-d-I-cis,trans 2,2-dimethyl-3-(2-methylpropenyl) cyclopropane carboxylate (phenothrin, CAS RN 26002-80-2); (empenthrin, CAS RN 54406-48-3); (cyphenothrin; CAS RN 39515-40-7), (prallethrin, CAS RN 23031-36-9), (imiprothrin, CAS RN 72963-72-5), (RS)-3-allyl-2-methyl-4-oxcyclopent-2-enyl-(1A,3R; 1R,3S)-2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropane carboxylate (allethrin, CAS RN 584-79-2), (bioallethrin, CAS RN 584-79-2), and (ZXI8901, CAS RN 160791-64-0). It is believed that mixtures of one or more of the aforementioned synthetic pyrethroids can also be used in the present invention. Particularly preferred synthetic pyrethroids are tefluthrin, lambda cyhalothrin, bifenthrin, permethrin and cyfluthrin. Even more preferred synthetic pyrethroids are tefluthrin and lambda cyhalothrin, and yet more preferred is tefluthrin.
Insecticides that are oxadiazine derivatives are useful in the subject method. The oxadizine derivatives that are preferred for use in the present invention include 5-(2-chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine, 3-methyl-4-nitroimino-5-(1-oxido -3-pyridinomethyl) perhydro-1,3,5-oxadiazine, 5-(2-chloro-1-oxido-5-pyridiniomethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxidiazine; and 3-methyl-5-(2-methylpyrid-5-ylmethyl)-4-nitroiminoperhydro-1,3,5-oxadiazine.
Chloronicotinyl insecticides are also useful in the subject method. Chloronicotinyls that are preferred for use in the subject composition include acetamiprid ((E)-N-[(6-chloro-3-pyridinyl)methyl]-Nxe2x80x2-cyano-N-methyleneimidamide, CAS RN 135410-20-7), imidacloprid (1-[(6-chloro-3-pyridinyl)methol]-N-nitro-2-imidazolidinimime, CAS RN 138261-41-3), and nitenpyram (N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-Nxe2x80x2-methyl-2-nitro-1,1-ethenediamine, CAS RN 120738-89-8).
Nitroguanidine insecticides are useful in the present method. Such nitroguanidines can include MTI 446 (nidinotefuran).
Pyrrols, pyrazoles and phenyl pyrazoles that are useful in the present method include those that are described in U.S. Pat. No. 5,952,358. Preferred pyrazoles include chlorfenapyr (4-bromo-2-(4-chlorophenyl)-1-ethoxymethyl-5-trifluoromethylpyrrole-3-carbonitrile, CAS RN 122453-73-0), fenpyroximate ((E)-1,1-dimethylethyl-4[[[[(1,3-dimethyl-5-phenoxy-1H-pyrazole-4-yl)methylene]amino]oxy]methyl]benzoate, CAS RN 111812-58-9), and tebufenpyrad (4-chloro-N[[4-1,1-dimethylethyl)phenyl]methyl]-3-ethyl-1-methyl-1H-pyrazole-5-carboxamide, CAS RN 119168-77-3). A preferred phenyl pyrazole is fipronil (5-amino-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(1R, S)-(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile, CAS RN 120068-37-3).
Diacylhydrazines that are useful in the present invention include halofenozide (4-chlorobenzoate-2-benzoyl-2-(1,1-dimethylethyl)-hydrazide, CAS RN 112226-61-6), methoxyfenozide (RH-2485; N-tert-buty-Nxe2x80x2-(3-methoxy-o-toluoyl)-3,5-xylohydrazide, CAS RN 161050-58-4), and tebufenozide (3,5-dimethylbenzoic acid 1-(1,1-dimethylethyl)-2,(4-ethylbenzoyl)hydrazide, CAS RN 112410-23-8).
Triazoles, such as amitrole (CAS RN 61-82-5) and triazamate are useful in the method of the present invention. A preferred triazole is triazamate (ethyl [[1-[(dimethylamino)carbonyl]-3-(1,1-dimethylethyl) -1H-1,2,4-triazol-5-yl]thio]acetate, CAS RN 112143-82-5).
Biological/fermentation products, such as avermectin (abamectin, CAS RN 71751-41-2) and spinosad (XDE-105, CAS RN 131929-60-7) are useful in the present method.
Organophosphate insecticides are also useful as one of the components of the present method. Preferred organophophate insecticides include acephate (CAS RN 30560-19-1), chlorpyrifos (CAS RN 2921-88-2), chlorpyrifos-methyl (CAS RN 5598-13-0), diazinon (CAS RN 333-41-5), fenamiphos (CAS RN 22224-92-6), and malathion (CAS RN 121-75-5).
In addition, carbamate insecticides are useful in the subject method. Preferred carbamate insecticides are aldicarb (CAS RN 116-06-3), carbaryl (CAS RN 63-25-2), carbofuran (CAS RN 1563-66-2), oxamyl (CAS RN 23135-22-0) and thiodicarb (CAS RN 59669-26-0).
When an insecticide is described herein, it is to be understood that the description is intended to include salt forms of the insecticide as well as any isomeric and/or tautomeric form of the insecticide that exhibits the same insecticidal activity as the form of the insecticide that is described.
The insecticides that are useful in the present method can be of any grade or purity that pass in the trade as such insecticide. Other materials that accompany the insecticides in commercial preparations as impurities can be tolerated in the subject methods and compositions, as long as such other materials do not destabilize the composition or significantly reduce or destroy the activity of any of the insecticide components or the transgenic event against the target pest(s). One of ordinary skill in the art of the production of insecticides can readily identify those impurities that can be tolerated and those that cannot.
It has been found that the present method is useful to protect seeds and plants against a wide array of agricultural pests, including insects, mites, fungi, yeasts, molds and bacteria.
When an insect is the target pest for the present invention, such pests include but are not limited to:
from the order Lepidoptera, for example,
Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae,Amylois spp., Anticarsia gemmatalis, Archips spp, Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia Nubilalis, Pammene spp., Pandemis spp., Panolis flammea, Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusia ni and Yponomeuta spp.;
from the order Coleoptera, for example,
Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinus spp., Popillia spp., Psylliodes spp., Rhizopertha spp., Scarabeidae, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.;
from the order Orthoptera, for example,
Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta ssp., and Schistocerca spp.;
from the order Isoptera, for example,
Reticulitemes ssp;
from the order Psocoptera, for example,
Liposcelis spp.;
from the order Anoplura, for example,
Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.;
from the order Mallophaga, for example,
Damalinea spp. and Trichodectes spp.; from the order Thysanoptera, for example,
Franklinella spp., Hercinothrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii;
from the order Heteroptera, for example,
Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Triatoma spp.;
from the order Homoptera, for example,
Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphax spp., Lacanium corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nehotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla ssp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae and Unaspis citri;
from the order Hymenoptera, for example,
Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius sppp., Monomorium pharaonis, Neodiprion spp, Solenopsis spp. and Vespa ssp.;
from the order Diptera, for example,
Aedes spp., Antherigona soccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp., Drosophila melanogaster, Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomysa spp., Lucilia spp., Melanagromyza spp., Musca ssp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp.,
from the order Siphonaptera, for example,
Ceratophyllus spp. und Xenopsylla cheopis and
from the order Thysanura, for example,
Lepisma saccharine.
It has been found that the present invention is particularly effective when the insect pest is a Diabrotica spp., and especially when the pest is Diabrotica virgifera, Diabrotica barberi, or Diabrotica undecimpunctata.
Another application wherein the present invention is believed to be particularly effective is when the pesticide has activity against a weed or a parasitic or saprophytic plant and the transgenic event has activity against a member selected from the group consisting of Diabrotica virgifera, Diabrotica barberi and Diabrotica undecimpunctata. This is believed to be more preferred useful when the weed or a parasitic or saprophytic plant is the African plant known as xe2x80x9cStrigaxe2x80x9d, and even more preferred when the pesticide is ROUNDUP(copyright) (available from Monsanto Company).
In the method of the present invention, the pesticide is applied to a transgenic corn seed. Although it is believed that the present method can be applied to a transgenic corn seed in any physiological state, it is preferred that the seed be in a sufficiently durable state that it incurs no damage during the treatment process. Typically, the seed would be a seed that had been harvested from the field; removed from the plant; and separated from any cob, stalk, outer husk, and surrounding pulp or other non-seed plant material. The seed would preferably also be biologically stable to the extent that the treatment would cause no biological damage to the seed. In one embodiment, for example, the treatment can be applied to seed corn that has been harvested, cleaned and dried to a moisture content below about 15% by weight. In an alternative embodiment, the seed can be one that has been dried and then primed with water and/or another material and then re-dried before or during the treatment with the pesticide. Within the limitations just described, it is believed that the treatment can be applied to the seed at any time between harvest of the seed and sowing of the seed. As used herein, the term xe2x80x9cunsown seedxe2x80x9d is meant to include seed at any period between the harvest of the seed and the sowing of the seed in the ground for the purpose of germination and growth of the plant.
When it is said that unsown seed is xe2x80x9ctreatedxe2x80x9d with the pesticide, such treatment is not meant to include those practices in which the pesticide is applied to the soil, rather than to the seed. For example, such treatments as the application of the pesticide in bands, xe2x80x9cTxe2x80x9d-bands, or in-furrow, at the same time as the seed is sowed are not considered to be included in the present invention.
The pesticide, or combination of pesticides, can be applied xe2x80x9cneatxe2x80x9d, that is, without any diluting or additional components present. However, the pesticide is typically applied to the seeds in the form of a pesticide formulation. This formulation may contain one or more other desirable components including but not limited to liquid diluents, binders to serve as a matrix for the pesticide, fillers for protecting the seeds during stress conditions, and plasticizers to improve flexibility, adhesion and/or spreadability of the coating. In addition, for oily pesticide formulations containing little or no filler, it may be desirable to add to the formulation drying agents such as calcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earth or any other adsorbent material. Use of such components in seed treatments is known in the art. See, e.g., U.S. Pat. No. 5,876,739. The skilled artisan can readily select desirable components to use in the pesticide formulation depending on the seed type to be treated and the particular pesticide that is selected. In addition, readily available commercial formulations of known pesticides may be used, as demonstrated in the examples below.
The seeds may also be treated with one or more of the following ingredients: other pesticides, including compounds which act only below the ground; fungicides, such as captan, thiram, metalaxyl, (methoxam=resolved isomer of metalaxyl), fludioxonil, oxadixyl, and isomers of each of those materials, and the like; herbicides, including compounds selected from carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxys, ureas, and benzoic acids; herbicidal safeners such as benzoxazine, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl and thiazolidinyl compounds, ethanone, naphthalic anhydride compounds, and oxime derivatives; fertilizers; and biocontrol agents such as naturally-occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These ingredients may be added as a separate layer on the seed or alternatively may be added as part of the pesticide composition.
Preferably, the amount of the novel composition or other ingredients used in the seed treatment should not inhibit generation of the seed, or cause phytotoxic damage to the seed.
The pesticide formulation that is used to treat the transgenic corn seed in the present invention can be in the form of a suspension; emulsion; slurry of particles in an aqueous medium (e.g., water); wettable powder; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation is preferably about 0.5% to about 99% by weight (w/w), preferably 5-40%.
As mentioned above, other conventional inactive or inert ingredients can be incorporated into the formulation. Such inert ingredients include but are not limited to: conventional sticking agents, dispersing agents such as methylcellulose (Methocel A15LV or Methocel A15C, for example, serve as combined dispersant/sticking agents for use in seed treatments), polyvinyl alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P), polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPNA S-630), thickeners (e.g., clay thickeners such as Van Gel B to improve viscosity and reduce settling of particle suspensions), emulsion stabilizers, surfactants, antifreeze compounds (e.g., urea), dyes, colorants, and the like. Further inert ingredients useful in the present invention can be found in McCutcheon""s, vol. 1, xe2x80x9cEmulsifiers and Detergents,xe2x80x9d MC Publishing Company, Glen Rock, N.J., U.S.A., 1996. Additional inert ingredients useful in the present invention can be found in McCutcheon""s, vol. 2, xe2x80x9cFunctional Materials,xe2x80x9d MC Publishing Company, Glen Rock, N.J., U.S.A., 1996.
The pesticides and pesticide formulations of the present invention can be applied to seeds by any standard seed treatment methodology, including but not limited to mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, and immersion. Any conventional active or inert material can be used for contacting seeds with pesticides according to the present invention, such as conventional film-coating materials including but not limited to water-based film coating materials such as Sepiret (Seppic, Inc., Fairfield, N.J.) and Opacoat (Berwind Pharm. Services, Westpoint, Pa.).
The subject pesticides can be applied to a seed as a component of a seed coating. Seed coating methods and compositions that are known in the art are useful when they are modified by the addition of one of the embodiments of the combination of pesticides of the present invention. Such coating methods and apparatus for their application are disclosed in, for example, U.S. Pat. Nos. 5,918,413, 5,891,246, 5,554,445, 5,389,399, 5,107,787, 5,080,925, 4,759,945 and 4,465,017. Seed coating compositions are disclosed, for example, in U.S. Pat. Nos. 5,939,356, 5,882,713, 5,876,739, 5,849,320, 5,834,447, 5,791,084, 5,661,103, 5,622,003, 5,580,544, 5,328,942, 5,300,127, 4,735,015, 4,634,587, 4,383,391, 4,372,080, 4,339,456, 4,272,417 and 4,245,432, among others.
Useful seed coatings contain one or more binders and at least one of the subject combinations of pesticides.
Binders that are useful in the present invention preferably comprise an adhesive polymer that may be natural or synthetic and is without phytotoxic effect on the seed to be coated. The binder may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; fats; oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.
It is preferred that the binder be selected so that it can serve as a matrix for the subject pesticides. While the binders disclosed above may all be useful as a matrix, the specific binder will depend upon the properties of the combination of pesticides. The term xe2x80x9cmatrixxe2x80x9d, as used herein, means a continuous solid phase of one or more binder compounds throughout which is distributed as a discontinuous phase one or more of the subject pesticides. Optionally, a filler and/or other components can also be present in the matrix. The term matrix is to be understood to include what may be viewed as a matrix system, a reservoir system or a microencapsulated system. In general, a matrix system consists of pesticides of the present invention and filler uniformly dispersed within a polymer, while a reservoir system consists of a separate phase comprising the subject pesticides, that is physically dispersed within a surrounding, rate-limiting, polymeric phase. Microencapsulation includes the coating of small particles or droplets of liquid, but also to dispersions in a solid matrix.
The amount of binder in the coating can vary, but will be in the range of about 0.01 to about 25% of the weight of the seed, more preferably from about 0.05 to about 15%, and even more preferably from about 0.1% to about 10%.
As mentioned above, the matrix can optionally include a filler. The filler can be an absorbent or an inert filler, such as are known in the art, and may include woodflours, clays, activated carbon, sugars, diatomaceous earth, cereal flours, fine-grain inorganic solids, calcium carbonate, and the like. Clays and inorganic solids, which may be used, include calcium bentonite, kaolin, china clay, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite and mixtures thereof. Sugars, which may be useful, include dextrin and maltodextrin. Cereal flours include wheat flour, oat flour and barley flour.
The filler is selected so that it will provide a proper microclimate for the seed, for example the filler is used to increase the loading rate of the active ingredients and to adjust the control-release of the active ingredients. The filler can aid in the production or process of coating the seed. The amount of filler can vary, but generally the weight of the filler components will be in the range of about 0.05 to about 75% of the seed weight, more preferably about 0.1 to about 50%, and even more preferably about 0.5% to 15%.
The pesticides that are useful in the coating are those pesticides that are described herein. The amount of pesticide that is used for the treatment of the seed will vary depending upon the type of seed and the type of active ingredients, but the treatment will comprise contacting the seeds with an amount of the combination of pesticides that is pesticidally effective. When insects are the target pest, that amount will be an amount of the insecticide that is insecticidally effective. As used herein, an insecticidally effective amount means that amount of insecticide that will kill insect pests in the larvae or pupal state of growth, or will consistently reduce or retard the amount of damage produced by insect pests.
In general, the amount of pesticide that is applied to the seed in the treatment will range from about 10 gm to about 2000 gm of the active ingredient of the pesticide per 100 kg of the weight of the seed. Preferably, the amount of pesticide will be within the range of about 50 gm to about 1000 gm active per 100 kg of seed, more preferably within the range of about 100 gm to about 600 gm active per 100 kg of seed, and even more preferably within the range of about 200 gm to about 500 gm of active per 100 kg of seed weight. Alternatively, it has been found to be preferred that the amount of the pesticide be over about 60 gm of the active ingredient of the pesticide per 100 kg of the seed, and more preferably over about 80 gm per 100 kg of seed.
In preferred embodiments of the present invention the transgenic event comprises the ability to express a Cry3Bb. 11231 protein or a Cry3Bb.11098 protein, and the pesticide is selected from either imidacloprid or tefluthrin.
The pesticides that are used in the treatment must not inhibit germination of the seed and should be efficacious in protecting the seed and/or the plant during that time in the target insect""s life cycle in which it causes injury to the seed or plant. In general, the coating will be efficacious for approximately 0 to 120 days after sowing.
The pesticides of the subject invention can be applied to the seed in the form of a coating. The use of a coating is particularly effective in accommodating high pesticidal loads, as can be required to treat typically refractory pests, such as corn rootworm, while at the same time preventing unacceptable phytotoxicity due to the increased pesticidal load.
Optionally, a plasticizer can be used in the coating formulation. Plasticizers are typically used to make the film that is formed by the coating layer more flexible, to improve adhesion and spreadability, and to improve the speed of processing. Improved film flexibility is important to minimize chipping, breakage or flaking during storage, handling or sowing processes. Many plasticizers may be used, however, useful plasticizers include polyethylene glycol, glycerol, butylbenzylphthalate, glycol benzoates and related compounds. The range of plasticizer in the coating layer will be in the range of from bout 0.1 to about 20% by weight.
When the pesticide used in the coating is an oily type formulation and little or no filler is present, it may be useful to hasten the drying process by drying the formulation. This optional step may be accomplished by means will known in the art and can include the addition of calcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earth, or any absorbent material that is added preferably concurrently with the pesticidal coating layer to absorb the oil or excess moisture. The amount of calcium carbonate or related compounds necessary to effectively provide a dry coating will be in the range of about 0.5 to about 10% of the weight of the seed.
The coatings formed with the pesticide are preferably of the type that are capable of effecting a slow rate of release of the pesticide by diffusion or movement through the matrix to the surrounding medium.
In addition to the coating layer, the seed may be treated with one or more of the following ingredients: other pesticides including fungicides and herbicides; herbicidal safeners; fertilizers and/or biocontrol agents. These ingredients may be added as a separate layer or alternatively may be added in the pesticidal coating layer.
The pesticide formulation may be applied to the seeds using conventional coating techniques and machines, such as fluidized bed techniques, the roller mill method, rotostatic seed treaters, and drum coaters. Other methods, such as spouted beds may also be useful. The seeds may be presized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.
The pesticide-treated seeds may also be enveloped with a film overcoating to protect the pesticide coating. Such overcoatings are known in the art and may be applied using conventional fluidized bed and drum film coating techniques.
In another embodiment of the present invention, a pesticide can be introduced onto or into a seed by use of solid matrix priming. For example, a quantity of the pesticide can be mixed with a solid matrix material and then the seed can be placed into contact with the solid matrix material for a period to allow the pesticide to be introduced to the seed. The seed can then optionally be separated from the solid matrix material and stored or used, or the mixture of solid matrix material plus seed can be stored or planted directly. Solid matrix materials which are useful in the present invention include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the pesticide for a time and releasing that pesticide into or onto the seed. It is useful to make sure that the pesticide and the solid matrix material are compatible with each other. For example, the solid matrix material should be chosen so that it can release the pesticide at a reasonable rate, for example over a period of minutes, hours, or days.
The present invention further embodies imbibition as another method of treating seed with the pesticide. For example, plant seed can be combined for a period of time with a solution comprising from about 1% by weight to about 75% by weight of the pesticide in a solvent such as water. Preferably the concentration of the solution is from about 5% by weight to about 50% by weight, more preferably from about 10% by weight to about 25% by weight. During the period that the seed is combined with the solution, the seed takes up (imbibes) a portion of the pesticide. Optionally, the mixture of plant seed and solution can be agitated, for example by shaking, rolling, tumbling, or other means. After imbibition, the seed can be separated from the solution and optionally dried, for example by patting or air drying.
In yet another embodiment, a powdered pesticide can be mixed directly with seed. Optionally, a sticking agent can be used to adhere the powder to the seed surface. For example, a quantity of seed can be mixed with a sticking agent and optionally agitated to encourage uniform coating of the seed with the sticking agent. The seed coated with the sticking agent can then be mixed with the powdered pesticide. The mixture can be agitated, for example by tumbling, to encourage contact of the sticking agent with the powdered pesticide, thereby causing the powdered pesticide to stick to the seed.
The present invention also provides a transgenic corn seed that has been treated with a pesticide by the method described above.
The treated seeds of the present invention can be used for the propagation of corn plants in the same manner as conventional treated corn seed. The treated seeds can be stored, handled, sowed and tilled in the same manner as any other pesticide treated seed. Appropriate safety measures should be taken to limit contact of the treated seed with humans, food or feed materials, water and birds and wild or domestic animals.
Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.
The following examples describe preferred embodiments of the invention. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples. In the examples all percentages are given on a weight basis unless otherwise indicated.

Method for reducing pest damage to corn by treating transgenic corn seeds with pesticide